Identification and quantification of Rhizoctonia solani and R. oryzae using real-time polymerase chain reaction.
Rhizoctonia solani and R. oryzae are the principal causal agents of Rhizoctonia root rot in dryland cereal production systems of the Pacific Northwest. To facilitate the identification and quantification of these pathogens in agricultural samples, we developed SYBR Green I-based real-time quantitative-polymerase chain reaction (Q-PCR) assays specific to internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA of R. solani and R. oryzae. The assays were diagnostic for R. solani AG-2-1, AG-8, and AG-10, three genotypes of R. oryzae, and an AG-I-like binucleate Rhizoctonia species. Quantification was reproducible at or below a cycle threshold (Ct) of 33, or 2 to 10 fg of mycelial DNA from cultured fungi, 200 to 500 fg of pathogen DNA from root extracts, and 20 to 50 fg of pathogen DNA from soil extracts. However, pathogen DNA could be specifically detected in all types of extracts at about 100-fold below the quantification levels. Soils from Ritzville, WA, showing acute Rhizoctonia bare patch harbored 9.4 to 780 pg of R. solani AG-8 DNA per gram of soil.. Blastn, primer-template duplex stability, and phylogenetic analyses predicted that the Q-PCR assays will be diagnostic for isolates from Australia, Israel, Japan, and other countries.
Okubara
P A
PA
U.S. Department of Agriculture-Agriculture Research Service, Root Disease and Biological Control Research Unit, Pullman, Washington 99164-6430, USA. pokubara@wsu.edu
Schroeder
K L
KL
Paulitz
T C
TC
D016000:Cluster Analysis; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D010935:Plant Diseases / Q000382:microbiology*; D018517:Plant Roots / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012232:Rhizoctonia / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer